Evaluation of Heating and Irradiation Methods for Production of Purified Protein Derivative (PPD) of Mycobacterium Tuberculosis.

Tuberculin skin test, also known as the tuberculin or purified protein derivative (PPD) test, is an extensively applied diagnostic test for the detection of primary infection with Mycobacterium tuberculosis (Mtb). The production of PPD is accompanied by some difficulties that require a series of modifications in the production and purification processes. The present study aimed to determine the facilitation level of the manufacturing process by modifying evaluation methods for the production of PPD tuberculin. Mtb strains were cultured in Lowenstein-Jensen media, and the cultured strains were inoculated into the Dorset-Henley liquid medium by the biphasic medium of potato-Dorset-Henley. After incubation, flasks containing cultured strain were selected for bacterial inactivation, and the optimal gamma radiation dose(s) was determined. Tuberculoproteins were precipitated by ammonium sulfate (AS) and Trichloroacetic acid (TCA). Protein concentration was determined using the Bradford and Kjeldahl protein assay methods. Finally, the lymphocyte transformation test and potency test were performed. Based on the results, the Dorset-Henley liquid medium is suitable for the massive growth of the bacterium. The transferal of Mtb from solid to liquid medium was directly carried out without intermediate culture. It was found that during tuberculoprotein production, heating at 100°C for 3 h would be safe for killing mycobacterium. Furthermore, the simultaneous use of heating and gamma irradiation (8 kGgy) killed all of the mycobacteria, while doses of 1, 1.5, and 7 kGy decreased a significant number of bacterial cells. The results also indicated that the concentration of tuberculoprotein extracted by TCA precipitation method was higher than that obtained by AS precipitation. The tuberculoproteins which were produced by these two methods in the lymphocyte transformation test were not significantly different in terms of potency (P>0.05). Moreover, due to the high volume of produced protein, the protein measurement was more efficiently carried out by the Kjeldahl method, compared to the Bradford method. Finally, the results of the present study demonstrated that in addition to the novel approach of gamma irradiation, optimum methods are efficient and applicable in the production of PPD tuberculin.

A tuberculin skin test survey among healthcare workers in two public tertiary care hospitals in Bangladesh.

In Bangladesh, there is currently no data on the burden of latent TB infection (LTBI) amongst hospital healthcare workers (HCWs). This study aimed to determine the prevalence of LTBI and compare the prevalence among HCWs in two public tertiary care hospitals. Between September 2018 and August 2019, we conducted a cross-sectional study in two public tertiary care general hospitals. Using a survey and tuberculin skin test (TST), we assessed risk factors for LTBI, adjusting for known and plausible confounders. In addition, a facility assessment was undertaken to understand the implementation of relevant IPC measures. The prevalence of LTBI among HCWs was 42%. HCWs spent a median of 6 hours (SD = 1.76, IQR 2.00) per day and attended an average of 1.87 pulmonary TB patients per week. HCWs did not receive any TB IPC training, the wards lacked a symptom checklist to screen patients for TB, and no masks were available for coughing patients. Seventy-seven percent reportedly did not use any facial protection (masks or respirators) while caring for patients. In the multivariable model adjusting for hospital level clustering effect, TST positivity was significantly higher among HCWs aged 35-45 years (aOR1.36, 95% CI: 1.06-1.73) and with >3 years of service (aOR 1.67, 95% CI: 1.62-1.72). HCWs working in the medicine ward had 3.65 (95% CI: 2.20-6.05) times, and HCWs in the gynecology and obstetrics ward had 2.46 (95% CI: 1.42-4.27) times higher odds of TST positivity compared to HCWs working in administrative areas. This study identified high prevalence of LTBI among HCWs. This may be due to the level of exposure to pulmonary TB patients, and/or limited use of personal protective equipment along with poor implementation of TB IPC in the hospitals. Considering the high prevalence of LTBI, we recommend the national TB program consider providing preventative therapy to the HCWs as the high-risk group, and implement TB IPC in the hospitals.

Endoscopic ultrasound in diagnosis of esophageal tuberculosis: 10-year experience at a tertiary care center.

Definite diagnosis of esophageal tuberculosis (ET) requires isolation of tubercle bacilli, which is challenging in clinical practice. Difficulty in differentiating ET from other esophageal diseases may well result in a delay in diagnosis. The literature on utility of endoscopic ultrasound (EUS) in diagnosis of ET is insufficient. This study aims to evaluate the role of EUS morphology combined with EUS-guided tissue acquisition in the diagnosis of ET. Data of the 35 patients diagnosed with ET from January 2006 to October 2015 were retrospectively analyzed. After miniprobe and linear echoendoscopic visualization, either linear EUS-guided deep biopsy or EUS-guided fine needle aspiration was performed for tissue acquisition. Histocytopathological results showing caseous necrosis or acid fast bacilli (AFB) or epithelioid granuloma were considered diagnostic. Esophageal wall thickening or mass formation with disruption of the adventitia due to infiltration by adjacent mediastinal lymphadenopathy was typically observed under EUS. Tissue acquisition revealed epithelioid granuloma in 33 patients, caseous necrosis in 13, a positive AFB stain in 14, and nonspecific chronic inflammation in 2. Of the 35 patients, 33 (94.3%) with both characteristic EUS morphology and diagnostic histocytopathology were considered to have an EUS established diagnosis. The remaining two with only nonspecific chronic inflammation received empirical antitubercular chemotherapy based solely on EUS morphology. The two-year follow-up confirmed diagnosis of ET in all patients. While the final diagnosis of ET was based upon two-year follow-up of treatment response to antitubercular medication in addition to caseous necrosis/granuloma/positive-AFB stain revealed by EUS-guided tissue acquisition, an EUS-established diagnosis of ET and medical treatment with long-term follow-up is rational and practical compared with surgery or untreated follow-up.

Cribado de la tuberculosis / Tuberculosis screening

Hay pruebas de que el cribado universal de la infección tuberculosa latente (ITBL) en zonas de baja incidencia tiene un rendimiento pobre, un número elevado de falsos positivos y no es coste-efectivo. Hay evidencias de que el cribado de la ITBL en los grupos que tienen riesgo elevado de desarrollar una infección tuberculosa activa (ITBA) mejora el rendimiento de las pruebas diagnósticas, el balance beneficio-riesgo y debe formar parte de las estrategias para disminuir el impacto de la tuberculosis. Sobre el dilema de cuál de las tres estrategias (prueba cutánea de la tuberculina [PCT] sola, determinación de la liberación de interferón gamma [IGRA] solo o PCT seguida de IGRA) para el diagnóstico de la ITBL es la que ofrece mejor rendimiento, existe variabilidad tanto en las recomendaciones de las guías de práctica clínica (GPC) y las instituciones como en las conclusiones de los estudios sobre el tema, aunque la mayoría de las recomendaciones van en el mismo sentido que las que proponemos en este documento. La prueba cutánea de la tuberculina sigue siendo el test diagnóstico con mejor rendimiento para realizar el cribado de la infección tuberculosa latente en niños y adolescentes. Recomendaciones de PrevInfad (GRADE): Se recomienda no realizar el cribado universal de la infección tuberculosa latente en los niños y adolescentes de nuestro país. Se recomienda realizar el cribado de la infección tuberculosa latente en los niños y adolescentes que pertenecen a grupos de riesgo. Se sugiere el uso de la prueba cutánea de la tuberculina (PCT) como primera prueba para el cribado de la infección tuberculosa latente en niños y adolescentes de nuestro país. Se sugiere utilizar los IGRA para los niños y adolescentes de cinco o más años con PCT positiva y antecedente de vacunación con bacilo de Calmette-Guérin (BCG) para mejorar la especificidad de la prueba de cribado (AU)

There is evidence that latent tuberculosis infection (LTBI) screening in low incidence areas, has a poor efficiency, many false positives and is not cost-effective. There is evidence that LTBI screening in groups at high risk of developing active tuberculosis infection (ATBI) improves the efficiency of the diagnostic tests as well as the risk-benefit balance and should take part of the strategies to reduce tuberculosis impact. Tuberculin skin test is still the best performing diagnostic test in the screening for latent tuberculosis infection in children and adolescents. PrevInfad recommendations (GRADE): Universal screening of latent tuberculosis infection in children and adolescents of our country is not recommended. Universal screening of latent tuberculosis infection in high risk groups of children and adolescents is recommended. Tuberculin skin test (TST) as the first test for screening of latent tuberculosis infection in children and adolescents of our country is suggested. IGRA for children and adolescents five or more years old with a positive TST and history of BCG vaccination, is suggested in order to improve the screening test specificity (AU)

Tuberculosis infantil: presentación de 3 casos y discusión / Pediatric tuberculosis: case report and discussion

Se presentan tres casos de tuberculosis infantil. Los dos primeros casos son dos hermanos de 5 años y 20 meses de edad con tuberculosis pulmonar, contactos intradomiciliarios del caso índice, la madre de ambos afecta de tuberculosis pulmonar bacilífera, con antecedente de viaje hace 4 meses a su pais de origen (Marruecos); ambos diagnosticados por positividad de la prueba de tuberculina y estudio radiográfico. El tercero es una niña de 9 años afecta de artritis idiopática juvenil en tratamiento inmunosupresor que desarrolló tuberculosis miliar con afectación encefálica, diagnosticada inicialmente en contexto de estudio de adenopatía (AU)

Three cases of childhood tuberculosis are presented: first and second cases are two brothers of 5 years and 2.0 months old respectively with pulmonary tuberculosis, household contacts of the index case: their mother, who was affected by smear-positive pulmonary tuberculosis, four months after visiting their country of origin (Morocco); both brethren were diagnosed by positivity of the tuberculin test and radiographic study. The third case is a 9 ycar old girl, juvenile idiopathic arthritis patient treated With immunosuppressive therapy, who has developed miliary tuberculosis with meningoencephalic involvement and she was initially diagnosed by the study of an adenopathy (AU)

[Production and evaluation of a purified protein derivative from an Argentine strain of Mycobacterium avium subsp. paratuberculosis]. / Obtención y evaluación de un derivado proteico purificado de una cepa argentina de Mycobacterium avium subsp. paratuberculosis.

Purified Protein Derivatives (PPDs) are non-defined antigens prepared from mycobacteria cultures. They are usually employed to evaluate the specific cellular immune response both in animals and humans. Bovine and avian PPDs are usually employed as antigens in mycobacterial infections such as tuberculosis and paratuberculosis. Nevertheless, PPD from Mycobacterium avium subsp. paratuberculosis, (PPDj) is neither commonly used nor frequently available. However, PPD from Mycobacterium avium subsp. avium is in fact used. We aimed to obtain and evaluate the performance of a PPDj from a local isolate of MAP using the ãInterferon-release assay. The stimulation of ãInterferon-release was significantly different between infected and control cattle when this antigen, named PPDj-IB, was used. Stimulation in the infected animals was similar with both antigens (PPDa and PPDj-IB). However, some animals were positively stimulated with PPDj-IB and not with PPDa. We demonstrated by Western blot that two antigenic molecules, lipoarabinoman and APA/ModD antigen were differentially represented in both PPDs. This could explain the difference in stimulation induction of yIFN observed at individual level. Although PPDj-IB could not improve PPDa performance, we could easily produce an effective purified protein derivative for in vitro assays.

Obtención y evaluación de un derivado proteico purificado de una cepa argentina de Mycobacterium avium subsp. paratuberculosis / Production and evaluation of a purified protein derivative from an Argentine strain of Mycobacterium avium subsp. paratuberculosis (MAP)

Los derivados proteicos purificados (PPD) son mezclas antigénicas no definidas obtenidas de distintas micobacterias. Los PPD bovino (PPDb) y PPD aviar (PPDa) son los antígenos que se emplean para evaluar la respuesta inmunitaria celular en infecciones como tuberculosis y paratuberculosis en el bovino. El PPDa comercial se produce a partir de Mycobacterium avium subsp. avium, y no a partir de la subespecie paratuberculosis. En este trabajo se seleccionó una cepa local de Mycobacterium avium subsp. paratuberculosis cuyo patrón molecular por RFLP es el más frecuente entre los aislamientos de nuestro país que han sido estudiados, y a partir de esta, se obtuvo un derivado proteico purificado: PPDj-IB. Se emplearon tanto el PPDa comercial como el PPDj-IB como antígenos en la prueba de liberación de gamma-interferón en animales de un tambo con paratuberculosis y en animales control. Aun cuando ambos PPD fueron capaces de estimular diferencialmente la liberación de la citoquina en el tambo infectado (respecto de los tambos control), no hubo diferencias significativas en los niveles de estimulación producidos y solo dos animales fueron positivos mediante el empleo de PPDj-IB. A partir del análisis por Western blot se demostró que el contenido de lipoarabinomano y del antígeno Apa/ModD era distinto en los PDD evaluados. Estas diferencias podrían explicar, en parte, las diferencias en los niveles de estimulación en términos individuales. Si bien el empleo de PPDj-IB no mejoró significativamente los resultados de la prueba de liberación de ?IFN, es importante destacar que se logró producir en el laboratorio un PPD apto para su empleo en ensayos in vitro.

Purified Protein Derivatives (PPDs) are non-defined antigens prepared from mycobacteria cultures. They are usually employed to evaluate the specific cellular immune response both in animals and humans. Bovine and avian PPDs are usually employed as antigens in mycobacterial infections such as tuberculosis and paratuberculosis. Nevertheless, PPD from Mycobacterium avium subsp. paratuberculosis, (PPDj) is neither commonly used nor frequently available. However, PPD from Mycobacterium avium subsp. avium is in fact used. We aimed to obtain and evaluate the performance of a PPDj from a local isolate of MAP using the ãInterferon-release assay. The stimulation of ãInterferon-release was significantly different between infected and control cattle when this antigen, named PPDj-IB, was used. Stimulation in the infected animals was similar with both antigens (PPDa and PPDj-IB). However, some animals were positively stimulated with PPDj-IB and not with PPDa. We demonstrated by Western blot that two antigenic molecules, lipoarabinoman and APA/ModD antigen were differentially represented in both PPDs. This could explain the difference in stimulation induction of ?IFN observed at individual level. Although PPDj-IB could not improve PPDa performance, we could easily produce an effective purified protein derivative for in vitro assays.

Obtención y evaluación de un derivado proteico purificado de una cepa argentina de Mycobacterium avium subsp. paratuberculosis / Production and evaluation of a purified protein derivative from an Argentine strain of Mycobacterium avium subsp. paratuberculosis (MAP)

Los derivados proteicos purificados (PPD) son mezclas antigénicas no definidas obtenidas de distintas micobacterias. Los PPD bovino (PPDb) y PPD aviar (PPDa) son los antígenos que se emplean para evaluar la respuesta inmunitaria celular en infecciones como tuberculosis y paratuberculosis en el bovino. El PPDa comercial se produce a partir de Mycobacterium avium subsp. avium, y no a partir de la subespecie paratuberculosis. En este trabajo se seleccionó una cepa local de Mycobacterium avium subsp. paratuberculosis cuyo patrón molecular por RFLP es el más frecuente entre los aislamientos de nuestro país que han sido estudiados, y a partir de esta, se obtuvo un derivado proteico purificado: PPDj-IB. Se emplearon tanto el PPDa comercial como el PPDj-IB como antígenos en la prueba de liberación de gamma-interferón en animales de un tambo con paratuberculosis y en animales control. Aun cuando ambos PPD fueron capaces de estimular diferencialmente la liberación de la citoquina en el tambo infectado (respecto de los tambos control), no hubo diferencias significativas en los niveles de estimulación producidos y solo dos animales fueron positivos mediante el empleo de PPDj-IB. A partir del análisis por Western blot se demostró que el contenido de lipoarabinomano y del antígeno Apa/ModD era distinto en los PDD evaluados. Estas diferencias podrían explicar, en parte, las diferencias en los niveles de estimulación en términos individuales. Si bien el empleo de PPDj-IB no mejoró significativamente los resultados de la prueba de liberación de ?IFN, es importante destacar que se logró producir en el laboratorio un PPD apto para su empleo en ensayos in vitro.(AU)

Purified Protein Derivatives (PPDs) are non-defined antigens prepared from mycobacteria cultures. They are usually employed to evaluate the specific cellular immune response both in animals and humans. Bovine and avian PPDs are usually employed as antigens in mycobacterial infections such as tuberculosis and paratuberculosis. Nevertheless, PPD from Mycobacterium avium subsp. paratuberculosis, (PPDj) is neither commonly used nor frequently available. However, PPD from Mycobacterium avium subsp. avium is in fact used. We aimed to obtain and evaluate the performance of a PPDj from a local isolate of MAP using the ÒInterferon-release assay. The stimulation of ÒInterferon-release was significantly different between infected and control cattle when this antigen, named PPDj-IB, was used. Stimulation in the infected animals was similar with both antigens (PPDa and PPDj-IB). However, some animals were positively stimulated with PPDj-IB and not with PPDa. We demonstrated by Western blot that two antigenic molecules, lipoarabinoman and APA/ModD antigen were differentially represented in both PPDs. This could explain the difference in stimulation induction of ?IFN observed at individual level. Although PPDj-IB could not improve PPDa performance, we could easily produce an effective purified protein derivative for in vitro assays.(AU)

Prevalencia de infección tuberculosa latente en población inmigrante que ingresa en prisión / Prevalence of latent tuberculosis infection amongst immigrants entering prison

Objetivo: Estudiar la prevalencia de infección tuberculosa latente (ITL) y sus factores predictivos en población reclusa inmigrante. Métodos: Estudio prospectivo realizado en mayo y junio de 2009. Se realizó intradermorreacción de Mantoux (IDRM), considerándose positiva la induración >= 10 mm. Se recogen las variables: edad, origen, reincidencia, tiempo en España, consumo de heroína y/o cocaína, uso de drogas intravenosas e infección VIH. Se calcula la tasa de ITL y la tasa global de infección (ITL más antecedente de tuberculosis). Para estudiar factores predictivos, se realizó un análisis bivariante y multivariante mediante regresión logística. Resultados: Se estudiaron 152 varones inmigrantes al ingreso en prisión. Edad media: 31,9 años ± 7,8. El 37,3% consumidor de heroína y/o cocaína y el 7,5% usuarios de drogas por vía intravenosa (UDI). 12 tenían IDRM previa positiva y 6 antecedente de TB. Se realizó IDRM a 134, 63 con resultado positivo y 71 con resultado negativo. Tasa de ITL: 49,3%. Tasa global de infección: 53,3%. Bivariadamente, se asoció a la ITL: la reincidencia (67,4% vs 36,4% en primarios, p=0,001), la edad (76% en los >= 40 años vs 40,4% en menores de esa edad; p=0,002) y el consumo de heroína y/o cocaína (60% en consumidores vs 39,3% en no consumidores; p= 0,02). El análisis multivariante sólo confirmó la asociación con la edad (p=0,001; OR: 2,34, IC= 1,39-3,94). Conclusiones: La tasa de ITL en inmigrantes que ingresan en prisión es muy elevada. Se recomienda en todos un completo estudio, con especial dedicación a los más vulnerables como los inmigrantes de mayor edad(AU)

Objective: To study the prevalence of latent tuberculosis infection (LTBI) and the predictive factors amongst immigrants entering prison. Methods: prospective study conducted in May and June of 2009. The tuberculin skin test (TST) was performed, with induration of >= 10 mm being regarded as positive. Variables collected were: age, origin, number of incarcerations, length of time living in Spain, heroin and cocaine consumption, intravenous drug use and HIV infection. The rate of LTBI was calculated and the overall infection rate (ITL and history of TB). To study predictable factors, a bivariant and multivariant analysis were carried out using logistic regression. Results: 152 male immigrants. Average age: 31.9 years ± 7.8; 37.2% of them with heroin or cocaine consumption and 7.5% IDU. 12 patients were previously TST positive and 6 patients had history of TB. TST was performed on 134 people, 63 with positive results and 71 with negative ones. ITL rate: 49.3. Overall infection rate: 53.3%. Bivariate associated with LTBI: more than one incarceration (67.4% vs. 36.4% in primary, p=0.001), age (76% >= 40 vs. 40.4% under this age and heroin and cocaine consumption (60% consumers vs. 39.3% non consumers; p=0.02. Multivariate analysis only confirmed the association with age (p=0.001; OR: 2.34, IC=1.39-3.94). Conclusions: The LTBI rate amongst immigrants entering prison is very high. A complete study is recommended for all of them, with special attention being paid to the most vulnerable ones, such as older people(AU)

10 Años innovando en el tratamiento de la infección tuberculosa latente: comparación entre pautas estándar y pautas cortas en tratamiento directamente observado / 10 Years of innovation in the treatment of latent tuberculosis infection: a comparison between standard and short course therapies in directly observed therapy

Objetivos: El objetivo principal del estudio fue comparar la aceptación, adherencia, tolerancia y seguridad de varias pautas cortas para el tratamiento de la infección tuberculosa latente (TIT), frente a una estándar de 9 meses, en tratamiento directamente observado (TDO) y confrontarlas con resultados previos de una pauta estándar en tratamiento autoadministrado por el paciente. Pacientes y métodos: Estudio longitudinal retrospectivo realizado en un centro penitenciario de tamaño medio. El período de inclusión abarcó 10 años, de enero de 2000 a diciembre de 2009. Se utilizaron los criterios de inclusión y exclusión de los Centers for Disease Control and Prevention (CDC) y los recogidos en el Programa de Prevención y Control de la Tuberculosis en el Medio Penitenciario. Se utilizaron 4 pautas de TIT según la preferencia del paciente y posibles interacciones con otros tratamientos. La pauta incluía isoniazida (H) en dosis de 300 mg/dia 9 meses (9H), la pauta II rifampicina más pirazinamida durante 2 meses 2 veces por semana, (2R2Z2) la pauta III rifampicina más isoniazida durante 3 meses (3RH) y la pauta IV rifampicina durante 4 meses (4R). Se administró el tratamiento de forma estricta en TDO por el personal de enfermería. Resultados: Se incluyen 902 pacientes, aceptando el tratamiento 810 (89,80%), distribuidos de la siguiente forma: 400 en la pauta 9H, y 410 con las pautas cortas (316 en la pauta 2R2Z2, 82 en la pauta 3RH y 12 en la pauta 4R. No aceptaron el TIT 92 (10,20%) pacientes. Finalizaron el TIT 271 (67,75%) con 9H, y 314 (76,60%) con las pautas cortas. Finalizaron con 2R2Z2, 232 pacientes (73,42%), con 3RH 70 (85,40%) y 12 (100%) con 4R. No finalizan el TIT con la pauta 9H 129 (32,25%) pacientes por los siguientes motivos (63 por abandono voluntario, 35 por reacciones adversas, 26 por libertad o traslado, 2 por causa desconocida, 1 por enfermedad tuberculosa en un paciente VIH- y 1 por suicidio). No finalizan el TIT con las pautas cortas 96 (23,41%) pacientes, por los siguientes motivos (36 por abandono voluntario, 54 por reacciones adversas, 1 por libertad o traslado, 3 por causa desconocida, 1 por brote psicótico en enfermo psiquiátrico y 1 por hepatitis aguda no filiada). Se aprecian diferencias significativas en las tasas de finalización del TIT al comparar la pauta estándar 9H y las pautas cortas. Se observa una mayor probabilidad de finalización, estadísticamente significativa, con las pautas cortas: p: 0,006; Odds Ratio: 1.56 (LC95%: 1.14-2.12). Este diferencia en la finalización se debe a que la pauta 9H presenta un mayor número de abandonos voluntarios sin motivo aparente (p: 0.002; OR: 2.03 [1.30-3.15]) y un mayor número de abandonos por conducción a otro centro o libertad (p<0,0001; OR 30.22 [4.07-224.29]), sin encontrarse diferencias significativas en los abandonos por reacciones adversas entre la pauta 9H y el conjunto de pautas cortas. ...(AU)

Objectives: The main aim of the study is to compare the acceptance, adherence, tolerance and safety of short course therapies in comparison to a standard 9 month treatment for latent tuberculosis infection (LTBI) in directly observed therapy (DOT) and contrast this with previous results from a standard therapy in patient self-administered treatment. Materials and methods: Retrospective longitudinal study carried out at a medium sized prison. Period of inclusion covers 10 years, from January 2000 to December 2009. The Centers for Disease Control and Prevention (CDC) inclusion and exclusion criteria were used, as well as the ones included in the Program for Tuberculosis Prevention and Control in the Prison Environment. 4 LTBI therapies according to the preference of the patient and possible interactions with other treatments were utilised. Therapy I consisted of isoniazid (H) in doses of 300 mg/day for 9 months (9H), therapy II with rifampicin for 2 months, twice a week, (2R2Z2) therapy III with rifampicin and isoniazid for 3 months (3RH) and therapy IV with rifampicin for four months (4R). Treatment was administered under strict DOT conditions by nursing staff. Results: 902 patients were included, of which 810 accepted the treatment (89.90%), distributed as follows: 400 in the 9H therapy, and 410 with short course therapies (316 in the 2R2Z2, 82 in the 3RH therapy and 12 in the 4R therapy). 92 patients (10.20%) did not accept LTBI therapy, 271 patients (67.75%) concluded the LTBI treatment with 9H, and 314 (76.60%) with short courses. 232 patients (73.42%) concluded the 2R2Z2, 85.40% with the 3RH 70 therapy and 12 (100%) with the 4R treatment. 129 patients (32.25%) did not complete the LTBI 9H therapy (63 due to voluntary withdrawal, 35 due to adverse reactions, 26 for release or transfer, 2 for unknown reasons, 1 due to tuberculosis in a HIV- patient and 1 due to suicide). 96 patients (23.41%) did not conclude the short course therapies (36 due to voluntary withdrawal, 54 due to adverse reactions, 1 due to release or transfer, 3 for unknown reasons, 1 due to a psychotic episode, and 1 due to hepatitis of unknown aetiology). Significant differences could be discerned in the LTBI therapy conclusion rates when comparing the standard 9H and short course therapies. A greater, statistically significant, probability is observed with the short course therapies: p: 0.006; Odds Ration: 1.56 (LC95%: 1.14-2.12). ...(AU)

Prevalence of tuberculin skin test positivity in clinical population in New York City.

The prevalence of latent tuberculosis infection (LTBI) in the various populations of New York City (NYC), a city with a high density of non-US-born persons, is unknown. We examined the prevalence of TST positivity in patients who received a tuberculin skin test (TST) between 1/2002 and 8/2004 at any of 10 NYC health department chest centers. A positive TST was defined as an induration reaction to tuberculin of ≥10 mm. In the study population of 41,022 individuals, prevalence of TST positivity was 24.4% (95%CI = 24.0, 24.8); four times higher among non-US-born persons than US-born (39.5% vs. 8.8%, Prevalence ratio (PR) = 4.5; 95%CI = 4.4, 4.6). Prevalence of TST positivity increased with age in both US and non-US-born persons. Persons from countries with a TB case rate >100/100,000 population had higher prevalence of TST positivity (47% vs. ≤39%), even after controlling for BCG (PR = 1.3, 95%CI = 1.2, 1.4). These findings provide insight into current prevalence of TST positivity in many immigrant populations and will help both clinicians and health departments to target patients for LTBI treatment.

Identification of proteins from tuberculin purified protein derivative (PPD) by LC-MS/MS.

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis, however it is poorly defined. Most mycobacterial proteins are extensively denatured by the procedure employed in its preparation, which explains previous difficulties in identifying constituents from PPD to characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based characterization of PPD from several different sources by LC-MS/MS, which combines the solute separation power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identified among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%) but there was a preponderance of proteins involved in lipid metabolism. We identified a group of 21 proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This study provides a better understanding of the tuberculin PPD components leading to the identification of additional antigens useful as reagents for specific diagnosis of tuberculosis.

Mycobacterial purified protein derivatives stimulate innate immunity: Malawians show enhanced tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-10 responses compared to those of adolescents in the United Kingdom.

To investigate the role of innate immunity in variable efficacy of Mycobacterium bovis BCG vaccination in Malawi and the United Kingdom, we examined 24-h tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-10 responses to mycobacterial purified protein derivatives (PPDs). The rank order in stimulatory potency for different PPDs was the same for all three cytokines. Before vaccination Malawians made higher pro- and anti-inflammatory responses than did United Kingdom subjects. Fewer than 5% of United Kingdom subjects made IL-10 in response to any PPD, compared to 19 to 57% responders among Malawians. Priming for regulatory IL-10 may contribute to the smaller increase in gamma interferon responses in Malawians compared to United Kingdom subjects following BCG vaccination.

Quality controls and in vitro diagnostic efficiency of bovine PPD tuberculins.

Wide heterogeneity was shown among different batches of bovine protein purified derivative (PPD) tuberculin, as regards protein content and antigenic profile. These features were also investigated in pilot preparations of Mycobacterium bovis secreted antigens and PPD tuberculins. Under controlled conditions, widely different compositions were revealed as a function of the M. bovis strain and also of the time in culture, due to the transient expression of seemingly important clusters of antigens. Furthermore, these parameters could dramatically affect the efficacy of the above preparations in in vitro assays of cell-mediated immunity on M. bovis-infected cattle. The field exposure to mycobacteria of the avium/intracellular group could also influence the readout of such assays, results being in agreement with a bystander suppression model of the response to M. bovis antigens. Due to the above, practical suggestions are put forward to improve the composition of bovine PPD tuberculins and the relevant control procedures.

[The isolation of the antigenic substances of Mycobacterium tuberculosis and the characteristics of their basic chemical components]. / Vydilennia antyhennykh substantsii mikobakterii tuberkul'ozu ta kharakterystyka ïkh osnovnykh khimichnykh komponentiv.

Different methods of isolation were used when obtaining the antigen determinants of the tuberculosis mycobacteria (tuberculins, polysaccharide, phosphatide). Main chemical components of the antigens were investigated. Most effective and economic methods of obtaining the M. tuberculosis antigens substances were chosen. The using of the obtained antigens in the test systems for diagnostics of tuberculosis discussed.

A 12-kDa protein of Mycobacterium tuberculosis protects mice against experimental autoimmune encephalomyelitis. Protection in the absence of shared T cell epitopes with encephalitogenic proteins.

Mycobacterium tuberculosis (Mt), routinely used to promote the induction of autoimmune diseases, can also protect against their development. Recently, we demonstrated that purified protein derivative (PPD) is the major fraction of Mt that protects mice against the induction of experimental autoimmune encephalomyelitis (EAE). We have now ascribed the protective activity to a 12-kDa protein purified from PPD. Sequence identity between the first 17 amino acids of the 12-kDa PPD protein and the 10-kDa BCG-a protein of Mt suggested that these proteins are identical or closely related. However, in contrast to the 12-kDa PPD protein, the 10-kDa BCG-a protein did not protect against EAE, nor did it stimulate PPD-specific T cells, suggesting that the 12-kDa PPD protein and the 10-kDa BCG-a protein share some homology but are not identical. The protective activity of the 12-kDa PPD protein correlated with its ability to stimulate PPD-specific T cells. The significance of this correlation is not clear and the mechanism of protection was not fully elucidated. However, N-terminal sequence identity between the 12-kDa PPD protein and the 10-kDa BCG-a protein, which shares 43% homology with GroES stress protein, suggested that the 12-kDa PPD protein may also belong to the bacterial heat-shock protein (hsp) family. Thus, by analogy with protection against arthritis or diabetes by hsp65, the mechanism of protection could be based on shared T cell epitopes with the target self Ag. However, the 12-kDa PPD protein did not stimulate encephalitogenic T lymphocytes. Effective protection against EAE by the 12-kDa PPD protein, in the absence of a stimulatory effect on encephalitogenic T lymphocytes, suggests a potential use for this protein in the therapy of autoimmune diseases.

The tuberculin test.

Tuberculin tests in general use today rely on the response to intradermal injections of tuberculin with assessment of the injection site for swelling at 72 hours post injection. Estimates of the sensitivity of tuberculin tests range from 68-95% while specificity is estimated to be 96-99%. The sensitivity of the test is affected by the potency and dose of tuberculin administered, the interval post-infection, desensitisation, deliberate interference, post-partum immunosuppression and observer variation. Specificity is influenced by sensitisation as a result of exposure to M. avium, M. paratuberculosis and environmental mycobacteria and by skin tuberculosis.

The 38-kDa protein of Mycobacterium tuberculosis: a review.

This review illustrates that the 38-kDa protein is one of the most important antigens of Mycobacterium tuberculosis. It is actively secreted but partly attached to the surface of the mycobacterial cell by a lipid tail that may also be responsible for binding of carbohydrate to the protein. It is a major constituent of M. tuberculosis culture fluid after growth on the synthetic Sauton medium and occurs in bacille Calmette-Guérin in far lower concentrations. The protein induces B and T cell responses with high specificity for infection with M. tuberculosis and is a prime candidate for development of new diagnostic reagents for tuberculosis.

[Post-vaccination tuberculin response using PPD derived from BCG]. / Viragem tuberculínica pós-vacinal com PPD derivado de BCG.

The aim of this study was to compare the biological response obtained with two tuberculin tests--PPD-RT 23 and PPD derived from BCG--among children already vaccinated with the Calmette-Guérin bacillus. Results varied widely, and the differences observed were statistically significant. This shows the need for additional studies to assess the sensitivity, specificity and predictive value of both tests.